Heightened resistance to host type 1 interferons characterizes HIV-1 at transmission and after antiretroviral therapy interruption

Type 1 interferons (IFN-I) are potent innate antiviral effectors that constrain HIV-1 transmission. However, harnessing these cytokines for HIV-1 cure strategies has been hampered by an incomplete understanding of their antiviral activities at later stages of infection. Here, we characterized the IFN-I sensitivity of 500 clonally derived HIV-1 isolates from the plasma and CD4+ T cells of 26 individuals sampled longitudinally after transmission or after antiretroviral therapy (ART) and analytical treatment interruption. We determined the concentration of IFNα2 and IFNβ that reduced viral replication in vitro by 50% (IC50) and found consistent changes in the sensitivity of HIV-1 to IFN-I inhibition both across individuals and over time. Resistance of HIV-1 isolates to IFN-I was uniformly high during acute infection, decreased in all individuals in the first year after infection, was reacquired concomitant with CD4+ T cell loss, and remained elevated in individuals with accelerated disease. HIV-1 isolates obtained by viral outgrowth during suppressive ART were relatively IFN-I sensitive, resembling viruses circulating just before ART initiation. However, viruses that rebounded after treatment interruption displayed the highest degree of IFNα2 and IFNβ resistance observed at any time during the infection course. These findings indicate a dynamic interplay between host innate responses and the evolving HIV-1 quasispecies, with the relative contribution of IFN-I to HIV-1 control affected by both ART and analytical treatment interruption. Although elevated at transmission, host innate pressures are the highest during viral rebound, limiting the viruses that successfully become reactivated from latency to those that are IFN-I resistant

Nuclear Factor 90(NF90) targeted to TAR RNA inhibits transcriptional activation of HIV-1

<p>Abstract</p> <p>Background</p> <p>Examination of host cell-based inhibitors of HIV-1 transcription may be important for attenuating viral replication. We describe properties of a cellular double-stranded RNA binding protein with intrinsic affinity for HIV-1 TAR RNA that interferes with Tat/TAR interaction and inhibits viral gene expression.</p> <p>Results</p> <p>Utilizing TAR affinity fractionation, North-Western blotting, and mobility-shift assays, we show that the C-terminal variant of nuclear factor 90 (NF90ctv) with strong affinity for the TAR RNA, competes with Tat/TAR interaction <it>in vitro</it>. Analysis of the effect of NF90ctv-TAR RNA interaction <it>in vivo </it>showed significant inhibition of Tat-transactivation of HIV-1 LTR in cells expressing NF90ctv, as well as changes in histone H3 lysine-4 and lysine-9 methylation of HIV chromatin that are consistent with the epigenetic changes in transcriptionally repressed gene.</p> <p>Conclusion</p> <p>Structural integrity of the TAR element is crucial in HIV-1 gene expression. Our results show that perturbation Tat/TAR RNA interaction by the dsRNA binding protein is sufficient to inhibit transcriptional activation of HIV-1.</p

HIV latency and integration site placement in five cell-based models

BACKGROUND: HIV infection can be treated effectively with antiretroviral agents, but the persistence of a latent reservoir of integrated proviruses prevents eradication of HIV from infected individuals. The chromosomal environment of integrated proviruses has been proposed to influence HIV latency, but the determinants of transcriptional repression have not been fully clarified, and it is unclear whether the same molecular mechanisms drive latency in different cell culture models. RESULTS: Here we compare data from five different in vitro models of latency based on primary human T cells or a T cell line. Cells were infected in vitro and separated into fractions containing proviruses that were either expressed or silent/inducible, and integration site populations sequenced from each. We compared the locations of 6,252 expressed proviruses to those of 6,184 silent/inducible proviruses with respect to 140 forms of genomic annotation, many analyzed over chromosomal intervals of multiple lengths. A regularized logistic regression model linking proviral expression status to genomic features revealed no predictors of latency that performed better than chance, though several genomic features were significantly associated with proviral expression in individual models. Proviruses in the same chromosomal region did tend to share the same expressed or silent/inducible status if they were from the same cell culture model, but not if they were from different models. CONCLUSIONS: The silent/inducible phenotype appears to be associated with chromosomal position, but the molecular basis is not fully clarified and may differ among in vitro models of latency

Temporal transcriptional response to latency reversing agents identifies specific factors regulating HIV-1 viral transcriptional switch

Background: Latent HIV-1 reservoirs are identified as one of the major challenges to achieve HIV-1 cure. Currently available strategies are associated with wide variability in outcomes both in patients and CD4+ T cell models. This underlines the critical need to develop innovative strategies to predict and recognize ways that could result in better reactivation and eventual elimination of latent HIV-1 reservoirs. Results and discussion: In this study, we combined genome wide transcriptome datasets post activation with Systems Biology approach (Signaling and Dynamic Regulatory Events Miner, SDREM analyses) to reconstruct a dynamic signaling and regulatory network involved in reactivation mediated by specific activators using a latent cell line. This approach identified several critical regulators for each treatment, which were confirmed in follow-up validation studies using small molecule inhibitors. Results indicate that signaling pathways involving JNK and related factors as predicted by SDREM are essential for virus reactivation by suberoylanilide hydroxamic acid. ERK1/2 and NF-κB pathways have the foremost role in reactivation with prostratin and TNF-aα, respectively. JAK-STAT pathway has a central role in HIV-1 transcription. Additional evaluation, using other latent J-Lat cell clones and primary T cell model, also confirmed that many of the cellular factors associated with latency reversing agents are similar, though minor differences are identified. JAK-STAT and NF-κB related pathways are critical for reversal of HIV-1 latency in primary resting T cells. Conclusion: These results validate our combinatorial approach to predict the regulatory cellular factors and pathways responsible for HIV-1 reactivation in latent HIV-1 harboring cell line models. JAK-STAT have a role in reversal of latency in all the HIV-1 latency models tested, including primary CD4+ T cells, with additional cellular pathways such as NF-κB, JNK and ERK 1/2 that may have complementary role in reversal of HIV-1 latency

In vitro reactivation of latent HIV-1 by cytostatic bis (thiosemicarbazonate) gold(III) complexes

BACKGROUND : A number of cytostatic agents have been investigated for the ability to reactivate latent viral reservoirs, which is a major prerequisite for the eradication of HIV-1 infection. Two cytostatic bis(thiosemicarbazonate) gold(III) complexes (designated 1 and 2) were tested for this potential in the U1 latency model of HIV-1 infection. METHODS : Cell viability in the presence or absence of 1 and 2 was determined using a tetrazolium dye and evidence of reactivation was assessed by p24 antigen capture following exposure to a latency stimulant, phorbol myristate acetate (PMA) and or test compounds. The latency reactivation mechanism was explored by determining the effect of the complexes on protein kinase C (PKC), histone deacetylases (HDAC) and proinflammatory cytokine production. RESULTS : The CC50 of the complexes in U1 cells were 0.53 ± 0.12 μM for 1 and 1.0 ± 0.4 μM for 2. In the absence of PMA and at non toxic concentrations of 0.2 and 0.5 μM, 1 and 2 significantly (p ≤ 0.02) reactivated virus in U1 cells by 2.7 and 2.3 fold respectively. In comparison, a 2.6 fold increase (p = 0.03) in viral reactivation was observed for hydroxyurea (HU), which was used as a cytostatic and latent HIV reactivation control. Viral reactivation was absent for the complexes during co-stimulation with PMA indicating the lack of an additive effect between the chemicals as well as an absence of inhibition of PMA induced HIV reactivation but for HU inhibition of the stimulant’s activity was observed (p = 0.01). Complex 1 and 2 activated PKC activity by up to 32% (p < 0.05) but no significant inhibition of HDAC was observed. Increases in TNF-α levels suggested that the reactivation of virus by the complexes may have been due to contributions from the latter and the activation of PKC. CONCLUSION : The ethyl group structural difference between 1 and 2 seems to influence bioactivity with lower active concentrations of 1, suggesting that further structural modifications should improve specificity. The cytostatic effect of 1 and 2 and now HIV reactivation from a U1 latency model is consistent with that of the cytostatic agent, HU. These findings suggest that the complexes have a potential dual (cytostatic and reactivation) role in viral “activation/elimination”.AuTEK Biomed (Mintek and Harmony Gold),Technology Innovation Agency (TIA) and the University of Pretoria.http://www.biomedcentral.com/bmcinfectdis/hb201

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